Hello
I am trying to detect phospho-H2AX (Ser139) to measure DNA damage. However, it seems that I detect the exact same bands using total H2A.X antibody and phospho-H2AX (both antibodies from cell signaling)... So I am not sure what I am doing wrong. I tried blocking with both, milk and BSA. Milk looks a bit better decreasing the background. I started using nuclear extracts, and I loaded 10 ug per well, which seemed a bit too much as the signal was too strong. How many micrograms of protein extract do you usually use? in both, whole cell lysates and nuclear extracts. Do you generally use milk or BSA as blocking agent? Any other tips I should know?
Thanks!!
Laura
I don't know the conditions you use for electrophoretic separation. But I think that you cannot distiguish between H2Ax and its phosphorylated form based on electrophoretic mobility. It should be (nearly) the same for both.
Another point is that you take care to avoid any protein phosphatase in your sample. You can block it with vanadate for example.
Hi Laura!
H2AX is a low-weight protein (~15 kDa) so it may be difficult to differenciate the phosphorylated variant from the non-phosphorylated one in an SDS-PAGE; take this into account when choosing the acrylamide concentration in the gels.
Regarding the intensity of the bands, if your are obtaining a very strong signal, you may try to reduce the total proteins loaded per well, especially if you are using nuclear extracts. It also may depend on the treatment you applied to your cells, the more DNA-aggresive treatment, the nore intense gamma-H2AX you may expect. In basal conditions, though, you may expect vary faint gamma-H2AX band and a strong H2AX one. In my experience, ionizing radiation dose over 1-2 Gy cause high number of high-signal gamma-H2AX foci detected by immunocytochemistry (FITC immunofluorescence). This technique is useful if you are expecting low to moderate amount of DNA double strand breaks, you may try, otherwise, you may persevere with WB. For high radiation doses (5 to 7.5 Gy) we tryed incubating the cells (human lymphocytes) for 24 hours, and measuring the persisting Gamma-H2AX foci (DBS) which are the ones the DNA repair systems cannot rejoin and may lead to cell death.
Hope my comments are of some use to you and good luck.
Hi Laura,
p-H2AX antibody has to be used in Western blotting. Note that Western procedure for histons is a bit different from that for other proteins. First, p-H2AX indicates that there is DNA damage (double strand breaks) but it is not a confirmatory test. For that you have to do either DNA fragmentation assay or TUNEL. For p-H2AX you should not use milk. I use 30 micro protein for loading (testis sample). It is better to do immuno along with WB just to confirm your WB results.
Dear Laura
I have used pH2aX from (ADI-KAM- CC255, Enzo Life Sciences, diluted 1:4000) on whole cell lysate loading 20-30ug protein on 10% SDS gels. As you can see the dilution factor was 1:4000. It is possible that your antibody dilution is too concentrated as you mention that the signal is too bright. So i suggest you block with 5% milk for 1hour after transfer, use a primary antibody dilution of 1:4000 TBS/Tw shaking at 4oC overnight and secondary antibody of 1:3000 or 1:5000 in TBS/Twn for 1hr at RT. For detection make sure that you start with a chemistry that is not High sensitivity. Usually for detection with Cell Signal antibodies i would use LumiGLO first and then the more sensitive LumiGLO Reserve.
If you do not detect with LumiGLO (or the less sensitive chemistry) simply wash the membrane in TBS/Tw and use the more sensitive chemistry.
Hope this information helps and good luck with your experiment
Kind regards
Luke
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